Cubosomes functionalized with antibodies as a potential strategy for the treatment of HER2-positive breast cancer.

Breast cancers that overexpress human epidermal growth factor receptor 2 (HER2) have poor prognosis. Moreover, available chemotherapies cause numerous side effects due to poor selectivity. To advance more effective and safer therapies for HER2-positive breast cancer, we explored the fusion of drug delivery technology and immunotherapy. Our research led to the design of immunocubosomes loaded with panobinostat and functionalized with trastuzumab antibodies, enabling precise targeting of breast cancer cells that overexpress HER2. We characterised the nanostructure of cubosomes using small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS). Moreover, we confirmed the integrity of the trastuzumab antibodies on the immunocubosomes by Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, we found that panobinostat-loaded immunocubosomes were more cytotoxic, and in an uptake-dependant manner, towards a HER2-positive breast cancer cell line (SKBR3) compared to a cell line representing healthy cells (L929). These results support that the functionalization of cubosomes with antibodies enhances both the effectiveness of the loaded drug and its selectivity for targeting HER2-positive breast cancer cells.

ASH1L guards cis-regulatory elements against cyclobutane pyrimidine dimer induction.

The histone methyltransferase ASH1L, first discovered for its role in transcription, has been shown to accelerate the removal of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair. Previous reports demonstrated that CPD excision is most efficient at transcriptional regulatory elements, including enhancers, relative to other genomic sites. Therefore, we analyzed DNA damage maps in ASH1L-proficient and ASH1L-deficient cells to understand how ASH1L controls enhancer stability. This comparison showed that ASH1L protects enhancer sequences against the induction of CPDs besides stimulating repair activity. ASH1L reduces CPD formation at C-containing but not at TT dinucleotides, and no protection occurs against pyrimidine-(6,4)-pyrimidone photoproducts or cisplatin crosslinks. The diminished CPD induction extends to gene promoters but excludes retrotransposons. This guardian role against CPDs in regulatory elements is associated with the presence of H3K4me3 and H3K27ac histone marks, which are known to interact with the PHD and BRD motifs of ASH1L, respectively. Molecular dynamics simulations identified a DNA-binding AT hook of ASH1L that alters the distance and dihedral angle between neighboring C nucleotides to disfavor dimerization. The loss of this protection results in a higher frequency of C->T transitions at enhancers of skin cancers carrying ASH1L mutations compared to ASH1L-intact counterparts.

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes.

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription.

Physiologically-Based Pharmacokinetic Modeling of the Postbiotic Supplement Urolithin A Predicts its Bioavailability Is Orders of Magnitude Lower than Concentrations that Induce Toxicity, but also Neuroprotective Effects.

SCOPE: A range of health benefits are attributed to consuming urolithin A (UA), such as improved muscle health, anti-aging activity, and neuroprotection, whereas few studies raise possible adverse effects at high doses, including genotoxicity and estrogenic effects. Therefore, understanding UA bioactivity and safety depends on its pharmacokinetics. However, there is no physiologically-based pharmacokinetic (PBPK) model available for UA, thus limiting reliable assessment of effects observed from in vitro experimentation. METHODS AND RESULTS: We characterizes glucuronidation rates of UA by human S9 fractions. Partitioning and other physicochemical parameters are predicted using quantitative structure-activity relationship tools. Solubility and dissolution kinetics are determined experimentally. These parameters are used to construct a PBPK model, and results are compared with data from human intervention studies. We evaluates how different supplementation scenarios may influence UA plasma and tissue concentrations. Concentrations at which either toxic or beneficial effects are previously observed in vitro appear unlikely to be achieved in vivo. CONCLUSION: A first PBPK model for UA is established. It enables prediction of systemic UA concentrations and is critical for extrapolating in vitro results to in vivo uses. Results support the safety of UA, but also challenge the potential for readily achieving beneficial effects by postbiotic supplementation.

Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures.

Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations.

Dissection of Cancer Mutational Signatures with Individual Components of Cigarette Smoking.

Tobacco smoke delivers a complex mixture of hazardous and potentially hazardous chemicals. Some of these may induce the formation of DNA mutations, which increases the risk of various cancers that display characteristic patterns of accumulated mutations arising from the causative exposures. Tracking the contributions of individual mutagens to mutational signatures present in human cancers can help understand cancer etiology and advance disease prevention strategies. To characterize the potential contributions of individual constituents of tobacco smoke to tobacco exposure-associated mutational signatures, we first assessed the toxic potential of 13 tobacco-relevant compounds by determining their impact on the viability of a human bronchial lung epithelial cell line (BEAS-2B). Experimentally derived high-resolution mutational profiles were characterized for the seven most potent compounds by sequencing the genomes of clonally expanded mutants that arose after exposure to the individual chemicals. Analogous to the classification of mutagenic processes on the basis of signatures from human cancers, we extracted mutational signatures from the mutant clones. We confirmed the formation of previously characterized benzo[a]pyrene mutational signatures. Furthermore, we discovered three novel mutational signatures. The mutational signatures arising from benzo[a]pyrene and norharmane were similar to human lung cancer signatures attributed to tobacco smoking. However, the signatures arising from N-methyl-N'-nitro-N-nitrosoguanidine and 4-(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone were not directly related to known tobacco-linked mutational signatures from human cancers. This new data set expands the scope of the in vitro mutational signature catalog and advances understanding of how environmental agents mutate DNA.

Highly Adhesive Amyloid-Polyphenol Hydrogels for Cell Scaffolding.

Rationally designing microstructures of soft hydrogels for specific biological functionalization is a challenge in tissue engineering applications. A novel and affordable soft hydrogel scaffold is constructed here by incorporating polyphenol modules with lysozyme amyloid fibrils (Lys AFs) via non-covalent self-assembly. Embedded polyphenols not only trigger hydrogel formation but also determine gel behavior by regulating the polyphenol gallol density and complex ratio. The feasibility of using a polyphenol-Lys AF hydrogel as a biocompatible cell scaffold, which is conducive to cell proliferation and spreading, is also shown. Notably, introducing polyphenols imparts the corresponding hydrogels a superior cell bioadhesive efficiency without further biofunctional decoration and thus may be successfully employed in both healthy and cancer cell lines. Confocal laser scanning microscopy also reveals that the highly expressed integrin-mediated focal adhesions form due to stimulation of the polyphenol-AF composite hydrogel, direct cell adhesion, proliferation, and spreading. Overall, this work constitutes a significant step forward in creating highly adhesive tissue culture platforms for in vitro culture of different cell types and may greatly expand prospects for future biomaterial design and development.

Smartphone-based magneto-immunosensor on carbon black modified screen-printed electrodes for point-of-need detection of aflatoxin B1 in cereals.

Considering the complexities and speed of modern food chains, there is an increasing demand for point-of-need detection of food contaminants, particularly highly regulated chemicals and carcinogens such as aflatoxin B1. We report a user-friendly smartphone-based magneto-immunosensor on carbon black modified electrodes for point-of-need detection of aflatoxin B1 in cereals. For buffered analyte solutions and a corn extract sample, the assay demonstrated a low limit of detection of 13 and 24 pg/mL, respectively. The assay was also highly reproducible, exhibiting mean relative standard deviations of 3.7% and 4.0% for the buffered analyte and corn extract samples. The applicability of the assay was validated on the basis of EU guidelines and the detection capability was lower than or equal to 2 µg/kg, which is the EU maximum residue limit for aflatoxin B1 in cereals. False-positive and false-negative rates were less than 5%. Additionally, an open-source android application, AflaESense, was designed to provide a simple interface that displays the result in a traffic-light-type format, thus minimizing user training and time for data analysis. AflaESense was used for smartphone-based screening of spiked corn samples containing aflatoxin B1 (0.1, 2, and 10 ng/mL), and naturally contaminated corn containing 0.15 ng aflatoxin B1/mL. The measured values were in close agreement with spiked concentrations (r2 = 0.99), with recovery values ranging between 80 and 120%. Finally, contaminated samples correctly triggered a red alert while the non-contaminated samples led to the display of a green color of AflaESense. To the best of our knowledge, this is the first smartphone-based electrochemical system effective for screening samples for contamination with aflatoxin B1.

Conformation and Pairing Properties of an O6-Methyl-2'-deoxyguanosine-Directed Benzimidazole Nucleoside Analog in Duplex DNA.

O6-Methyl-2'-deoxyguanosine (O6-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by S-adenosylmethionine. O6-MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as O6-MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at O6-MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with O6-MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and O6-MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or O6-MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or O6-MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between O6-MeG and the extended π system of the synthetic probe.

A combination of direct reversion and nucleotide excision repair counters the mutagenic effects of DNA carboxymethylation.

Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O6-carboxymethylguanine (O6-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O6-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O6-CMG mutagenicity. To better address how cells detect and remove O6-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O6-CMG. We found that cells deficient in either the direct repair protein O6-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O6-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O6-CMG DNA damage.

Molecular Dosimetry of Temozolomide: Quantification of Critical Lesions, Correlation to Cell Death Responses, and Threshold Doses.

Temozolomide (TMZ) is a DNA-methylating agent used in cancer chemotherapy, notably for glioblastoma multiforme (GBM), where it is applied as a front-line drug. One of the DNA alkylation products of TMZ is the minor lesion O6 -methylguanine (O6 MeG), which is responsible for nearly all genotoxic, cytotoxic, and cytostatic effects induced in the low-dose range relevant for cancer therapy. Here, we addressed the question of how many O6 MeG adducts are required to elicit cytotoxic responses. Adduct quantification revealed that O6 MeG increases linearly with dose. The same was observed for DNA double-strand breaks (DSB) and p53ser15. Regarding apoptosis, hockeystick modeling indicated a possible threshold for A172 cells at 2.5 µmol/L TMZ, whereas for LN229 cells no threshold was detected. Cellular senescence, which is the main cellular response, also increased linearly, without a threshold. Using a dose of 20 µmol/L, which is achievable in a therapeutic setting, we determined that 14,000 adducts give rise to 32 DSBs (γH2AX foci) in A172 cells. This leads to 12% cell death and 35% of cells entering senescence. In LN229 cells, 20 µmol/L TMZ induced 20,600 O6 MeG adducts, 66 DSBs (γH2AX foci), 24% apoptosis, and 52% senescence. The linear dose response and the genotoxic and cytotoxic effects observed at therapeutically relevant dose levels make it very likely that the TMZ target concentration triggers a significant cytotoxic and cytostatic effect in vivo Despite a linear increase in the O6 MeG adduct level, DSBs, and p53 activation, the low curative effect of TMZ results presumably from the low rate of apoptosis compared to senescence.

Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells.

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.

Direct Alkylation of Deoxyguanosine by Azaserine Leads to O6-Carboxymethyldeoxyguanosine.

The O6-alkylguanosine adduct O6-carboxymethyldeoxyguanosine (O6-CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of O6-CMdG as well as the corresponding methyl adduct O6-methyldeoxyguanosine (O6-MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize O6-CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of O6-CMdG and O6-MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an O6-serine-CMdG (O6-Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O6-Ser-CMdG forms as an intermediate that spontaneously decomposes to form O6-CMdG. Finally, we quantified levels of O6-CMdG and O6-MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.

Impact of manipulation of glycerol/diol dehydratase activity on intestinal microbiota ecology and metabolism.

Glycerol/diol dehydratases (GDH) are enzymes that catalyse the production of propionate from 1,2-propanediol, and acrolein from glycerol. Acrolein reacts with dietary carcinogenic heterocyclic amines (HCA), reducing HCA mutagenicity, but is itself also an antimicrobial agent and toxicant. Gut microbial GDH activity has been suggested as an endogenous acrolein source; however, there is limited information on the potential of the intestinal microbiota to have GDH activity, and what impact it can have on the intestinal ecosystem and host health. We hypothesized that GDH activity of gut microbiota is determined by the abundance and distribution of GDH-active taxa and can be enhanced by supplementation of the GDH active Anaerobutyricum hallii, and tested this hypothesis combining quantitative profiling of gdh, model batch fermentations, microbiota manipulation, and kinetic modelling of acrolein formation. Our results suggest that GDH activity is a common trait of intestinal microbiota shared by a few taxa, which was dependent on overall gdh abundance. Anaerobutyricum hallii was identified as a key taxon in GDH metabolism, and its supplementation increased the rate of GDH activity and acrolein release, which enhanced the transformation of HCA and reduced fermentation activity. The findings of this first systematic study on acrolein release by intestinal microbiota indicate that dietary and microbial modulation might impact GDH activity, which may influence host health.

A Chemical Link between Meat Consumption and Colorectal Cancer Development?

O6-carboxymethylguanine (O6-CMG) is a mutagenic DNA adduct that forms at increased levels when people eat meat. It has been studied as a potential initiating event in colorectal carcinogenesis. It can arise from alkylation of guanine in DNA by electrophilic degradation products of N-nitroso compounds. There is significant data regarding biochemical and cellular process, including DNA repair and translesion DNA synthesis that control O6-CMG accumulation, persistence, and mutagenicity. Mutation spectra arising from the adduct closely resemble common mutations in colorectal cancer; however, gaps remain in understanding the biochemical processes that regulate how and where the damage persists in the genome. Addressing such questions relies on advances in chemistry such as synthesis approaches and bioanalytical methods. Results of research in this area help advance our understanding of the toxicological relevance of O6-CMG-modified DNA. Further attention should focus on understanding how a combination of genetic and environmental factors control its biological persistence and how this information can be used as a basis of biomoniotoring and prevention efforts to help mitigate colon cancer risk.

Next-generation DNA damage sequencing.

Cellular DNA is constantly chemically altered by exogenous and endogenous agents. As all processes of life depend on the transmission of the genetic information, multiple biological processes exist to ensure genome integrity. Chemically damaged DNA has been linked to cancer and aging, therefore it is of great interest to map DNA damage formation and repair to elucidate the distribution of damage on a genome-wide scale. While the low abundance and inability to enzymatically amplify DNA damage are obstacles to genome-wide sequencing, new developments in the last few years have enabled high-resolution mapping of damaged bases. Recently, a number of DNA damage sequencing library construction strategies coupled to new data analysis pipelines allowed the mapping of specific DNA damage formation and repair at high and single nucleotide resolution. Strikingly, these advancements revealed that the distribution of DNA damage is heavily influenced by chromatin states and the binding of transcription factors. In the last seven years, these novel approaches have revealed new genomic maps of DNA damage distribution in a variety of organisms as generated by diverse chemical and physical DNA insults; oxidative stress, chemotherapeutic drugs, environmental pollutants, and sun exposure. Preferred sequences for damage formation and repair have been elucidated, thus making it possible to identify persistent weak spots in the genome as locations predicted to be vulnerable for mutation. As such, sequencing DNA damage will have an immense impact on our ability to elucidate mechanisms of disease initiation, and to evaluate and predict the efficacy of chemotherapeutic drugs.

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide.

DNA mutations can result from replication errors due to different forms of DNA damage, including low-abundance DNA adducts induced by reactions with electrophiles. The lack of strategies to measure DNA adducts within genomic loci, however, limits our understanding of chemical mutagenesis. The use of artificial nucleotides incorporated opposite DNA adducts by engineered DNA polymerases offers a potential basis for site-specific detection of DNA adducts, but the availability of effective artificial nucleotides that insert opposite DNA adducts is extremely limited, and furthermore, there has been no report of a quantitative strategy for determining how much DNA alkylation occurs in a sequence of interest. In this work, we synthesized an artificial nucleotide triphosphate that is selectively inserted opposite O6-carboxymethyl-guanine DNA by an engineered polymerase and is required for DNA synthesis past the adduct. We characterized the mechanism of this enzymatic process and demonstrated that the artificial nucleotide is a marker for the presence and location in the genome of O6-carboxymethyl-guanine. Finally, we established a mass spectrometric method for quantifying the incorporated artificial nucleotide and obtained a linear relationship with the amount of O6-carboxymethyl-guanine in the target sequence. In this work, we present a strategy to identify, locate, and quantify a mutagenic DNA adduct, advancing tools for linking DNA alkylation to mutagenesis and for detecting DNA adducts in genes as potential diagnostic biomarkers for cancer prevention.

Synthesis of 4-Cyanoindole Nucleosides, 4-Cyanoindole-2'-Deoxyribonucleoside-5'-Triphosphate (4CIN-TP), and Enzymatic Incorporation of 4CIN-TP into DNA.

4-Cyanoindole-2'-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2'-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4-cyanoindole-ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase.